Thiamin is an essential component of the human diet with an RDA of 1.2 mg. It is also an important commercial chemical and it is widely used as a food additive and flavoring agent. Annual production, by chemical synthesis, is on the order of 3,300 tons. Thiamin-dependent enzymes play an important role in carbohydrate and branched-chain amino acid metabolism. At this time, our mechanistic understanding of this class of enzymes is sophisticated. In contrast, while thiamin was the first vitamin identified, our understanding of its biosynthesis is still incomplete because the biosynthetic pathway is complex and involves unprecedented reaction chemistry. Thiamin consists of a thiazole linked to a pyrimidine. The thiazole moiety in bacteria is formed from deoxy-D-xylulose-5-phosphate, glycine, and a 66 amino acid protein thiocarboxylate via a complex oxidative condensation. The pyrimidine in bacteria is formed from 5-aminoimidazole ribonucleotide via a complex rearrangement reaction. The pyrimidine is then coupled to the thiazole to give thiamin phosphate and a final phosphorylation gives the biologically active form of vitamin B1. Our mechanistic understanding of thiazole formation in bacteria is now at an advanced stage and we have recently established some of the main features of thiazole formation in eukaryotes. The pyrimidine thiazole coupling reaction is also well understood and the pyrimidine carbocation intermediate has been structurally and kinetically characterized. In contrast our mechanistic understanding of the remarkable chemistry involved in the formation of the thiamin pyrimidine in bacteria and in eukaryotes is still at an early stage. In the next funding period, we propose to continue our mechanistic characterization of the thiazole biosynthetic enzymes, evaluate the generality of the novel sulfur transfer chemistry involved in thiazole formation, and carry out mechanistic and structural studies on the pyrimidine biosynthetic enzymes. In addition, we will explore the enzymology of the salvage of acid-degraded thiamin. The long-term goal of our research is the complete mechanistic understanding of the enzymology of thiamin biosynthesis in both prokaryotes and eukaryotes and the elucidation of connections between thiamin metabolism and other aspects of cellular physiology. Our studies are significant for four reasons. First, it is important to understand how thiamin is biosynthesized because this vitamin is a required component of the human diet and an essential cofactor for all forms of life. Second, the biosynthetic pathway involves an unusually large amount of unprecedented biological chemistry. Third, our studies will facilitate the construction of overexpression strains that will be of use for the commercial production of thiamin by fermentation. Finally, inhibitors of thiamin biosynthetic enzymes may provide a selective strategy for antibiotic design in bacteria such as Mycobacterium tuberculosis that lack a thiamin transport system. PUBLIC HEALTH RELEVANCE We are interested in how vitamin B1 (thiamin) is assembled in living systems. This is an important problem because vitamin B1 is a required component of the human diet and is essential for all forms of life. Thiamin is added to many foods and our studies will facilitate its commercial production by fermentation. In addition, our research has potential applications in the design of TB specific antibiotics.